Background/Purpose: We have demonstrated that the degree of tubulointerstitial nephritis (TIN) on diagnostic biopsy predicts progression to renal failure. TIN is associated with tertiary lymphoid structures, in situ B cell oligoclonal expansion and ongoing antigen-driven somatic hypermutation. These phenomena support the hypothesis that local antigen is driving in situ lymphocyte selection and activation. Therefore, we cloned and characterized the antigenic specificities of in situ selected antibodies in human TIN.  Methods: Human kidney biopsies with lupus TIN were stained for the proliferation marker ki-67 (6/8), or the post GC marker CD38 (1/8). Positive single cells were isolated by laser capture microscopy. An eighth sample was single cell sorted for CD138⁺ plasmablasts. mRNA was purified and cDNA reverse transcribed. Matched IgG heavy and light chain variable regions were PCR amplified, cloned into human IgG1 heavy or light chain expression vectors and expressed as mAbs in HEK-293 cells. Antigen screening methods included clinical ELISAs, crithidia IIF (for DNA), human protein arrays (Invitrogen), confocal microscopy of HEp-2 cells. Antigens targeted in HEp-2 cells were sought by immunoprecipitation followed by mass spectrometry. Candidate antigen identity was confirmed by multi-color confocal microscopy, and purified antigen ELISAs and protein arrays. In situ expression pattern of target antigen was confirmed by immunofluorescence using kidney from lupus TIN and normal controls. Sera from lupus patients, with (n=40) and without nephritis (n=20), were tested for IgG reactivity with candidate antigens. Results: 27 mAbs were generated. By HEp-2 analysis, 3/27 yielded nuclear speckled-patterns, 1/27 nucleolar-, 7/27 cytoskeletal-, 3/27 nuclear and cytoplasmic-, and 1/27 reacted with the golgi apparatus. None were DNA, Sm or RNP positive. Vimentin was confirmed as the dominant targeted antigen (40% of mAbs, from 7/8 patients), immunoprecipitated from HEp-2 cells, bound by TIN mAbs on arrays and ELISA, and yielding a co-staining pattern with an anti-vimentin antibody (V9, DAKO). Vimentin reactive TIN mAbs reacted strongly with both glomeruli and inflamed TI, but little with normal TI. Approximately 70% of serum samples reacted with vimentin by protein array and this tended to be more common in nephritis patients. Antigens targeted more strongly in nephritic serum included myosin and SSB (median % false discovery rate <.001). Conclusion: Vimentin is an autoantigen upregulated during inflammation capable of driving in situ antibody production. These data support a model inwhich a positive feed-back loop of inflammation contributes to renal failure.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterisation-of-antigens-driving-in-situ-autoantibody-production-in-human-lupus-tubulointerstitial-nephritis-tin/